The mating of orthogonal, or complementary, methods of separating analytes of interest is of fundamental importance to the advancement of the study of highly complex systems. Such matings are commonplace in the art, however for each combination and respective order thereof, specific attributes are required of the mated instruments. Herein methodology for novel matings of analytical instrumentation are described.
Traditionally, ion mobility spectrometry (IMS) has been used for the analysis of gas phase compounds (Hill et al., Anal. Chem. 62(23):1201A, 1990; Eiceman and Karpas, Ion Mobility Spectrometry, CRC Press, New York, N.Y., p. 1-15, 1994). Moreover, with the advent of electrospray ionization (ESI) (Fenn et al., Science 246(4926):64-71, 1989), and matrix-assisted laser desorption ionization (MALDI) (Tanaka et al., Rapid Comm. In Mass Spec. 2(8):151-153, 1988; Karas and Hillenkamp, Anal. Chem. 60(20):2299-2301, 1988) the scope of IMS analysis has greatly expanded (Shumate, et al., Anal. Chem. 61:601-606, 1989; Helden et al., Science 267:1483-1485, 1995; Khalid, et al., Rapid Commun. In Mass Spectrometry, 17, 87-96, 2003).
In the presence of a neutral drift gas, IMS separates gas phase ions based upon their differential migration through a weak homogeneous electric field (Revercomb and Mason, Anal Chem. 47(7):970-983, 1975). In cases where the analyte ions are much larger than the drift gas molecule, an ion's mobility is proportional to its collision cross section, Ω, and charge, z. In these traditional ‘low-field’ applications a typical analyte resident time within a drift tube is on the order of ten's of milliseconds.
Newer implementations employ high-field IMS (‘high-field’ asymmetric waveform ion mobility spectrometry, FAIMS, or differential mobility spectrometry, DMS collectively referred to hereinafter as ‘High-Field’ IMS), the ions are separated on their interaction in a rapidly switched high and low electric field. In high-field IMS, the ions are pneumatically moved through a switching electric field which is orthogonal to the pneumatic flow. Under the high-field condition (˜10,000 V/cm) the ions are rapidly accelerated for a short time-constant toward the respective positive or negative electrode. After a few hundred nanoseconds, the high electric field is reversed to a low electric field of opposite polarity for twice the time-constant, forcing the ions to change their direction. Without further assistance, the ion's trajectory would have them collide with an electrode and never exit the system. Therefore, akin to the DC offset in a quadrupole mass spectrometer, the zero-crossing point of the switching voltages is slowly changed, or compensated, incrementally allowing ions to exit the system, yielding a compensation-voltage-dependent ion mobility spectrum. Even though the resulting spectrum is a function of the compensation voltage, separation occurs based on the difference of their response in the high and low electric fields. Within these high-field applications a typical analyte resident time within a drift tube is on the order of hundreds of milliseconds.
Since the high-and low-field techniques separate ions differently, it would naturally be beneficial to couple the instruments as has been successfully implemented in the art. The output from a high-field ion mobility spectrometer was directed to the input of a low-field ion mobility spectrometer instrument (Tang et al., Anal. Chem. 77:6381-6388, 2005). When coupling the low-field (and much faster) instrument to the output of the high-field (and slower) instrument, multiple low-field IMS scans can be performed in the time it takes for the ions to traverse the high-field system. Significantly, due to the discrepancies with the residence times, coupling the instruments in the reverse order (low-field output into high-field input) will cause loss of resolution from the low-field IMS. Since the low-field device processes analytes more rapidly the separation afforded by the low-field device will be undone by the backlog created at the entrance of the high-field device.
Another desirable combination of instrumentation is provided by the coupling of IMS instrumentation to mass spectrometers (MS). The IMS concept of measuring size-to-charge ratio is complementary to the principle of measurement in mass spectrometry (MS) of mass-to-charge ratio (m/z). When combined, ion mobility-mass spectrometry (IMMS or IM:MS) represents a powerful analytical combination capable of distinguishing ions based upon both size and mass to charge ratios. This union has provided an added degree of specificity above that attainable by mass spectrometry alone. While low pressure, low-field IMMS systems have been described in the art, (Clemmer, U.S. Pat. No. 6,559,441, Clemmer, U.S. Pat. No. 7,077,944, and Clemmer, U.S. Pat. No. 5,905,258) these systems employ MS methods (e.g., TOF-MS) wherein the timescale of MS sampling is much faster than the transit time of an analyte through the low-field IMS drift tube. These implementations highlight the larger problem one faces when attempting to couple orthogonal methods of analytical characterization: In order to obtain meaningful separations and characterizations, the timescale of the first method must be slower than the timescale of the second. High-field IMMS couplings are also common in the art and generally do not suffer from such timing problems. (Guevremont, et al. U.S. Pat. No. 6,504,149; Purves et al. Review of Scientific Instruments 69 (12):4094-4105, 1998).
While MS alone has achieved resolution (m/Δm50%) measurements of over 400,000 (Bossio et al., Anal. Chem. 74(7):1674-1679, 2002), routine differentiation between isomeric species using mass spectrometry has proven elusive. Often distinguishing features for a particular isomer require complex multidimentional mass spectroscopic (MSn) experiments where the measurement of relative peaks heights is necessary for identification (Tao et al., J. Am. Chem. Soc. 122(43):10598-10609, 2000; Leavell et al., J. Am. Soc. Mass Spectrom. 14(4):323-331, 2003). Because IMS can separate analytes based on their size to charge ratio, low-field IMMS has been employed to differentiate between isomeric species, IMMS experiments have demonstrated the ability to conclusively identify isomeric species (Wu et al., Anal. Chem. 72:391-395, 2000; Srebalus-Barnes et al., Anal. Chem. 74:26-36, 2002; Clowers et al., J. Am. Soc. Mass Spectrom. 16(5):660-669, 2005).
Hybrid low-field IMMS instruments have also been used to obtain conformational information for proteins (Sowell et al., J. Am. Soc. Mass Spectrom. 15:1341-1353, 2004; Ruotolo et al., Int. J. Mass Spec. 219(1):253-267, 2002; Bernstein et al., J. Am. Chem. Soc. 127:2075-2084, 2005), peptides (Srebalus-Barnes et al., Anal. Chem. 74:26-36, 2002; Hill et al., Int. J. Mass Spec. 219(1):23-37, 2002; Ruotolo et al., J. Prot. Res. 1(4):303-306, 2002; Barran et al., Int. J. Mass Spec. 240(3):274-284, 2005; Breaux and Jarrold, J. Am. Chem. Soc. 125:10740-10747, 2003), carbohydrates (Clowers et al., J. Am. Soc. Mass Spectrom. 16(5):660-669, 2005; Lee et al., J. Mass Spec. Ion Proc. 167/168:605-614, 1997; Leavell et al., J. Am. Soc. for Mass Spec. 13(3):284-293, 2002) and other biologically relevant system (Wyttenbach et al., Int. J. Mass Spec. 193:143-152, 1999; Koomen et al., Anal. Bioanal. Chem. 373(7):612-617, 2002; Gidden et al., Int. J. Mass Spec. 240:183-193, 2005; Matz et al., Anal. Chem. 73(8):1664-1669, 2001).
Mass spectrometry techniques that have successfully been interfaced with low-field ion mobility include: quadrupole (QMS), (Karasek et al., Anal. Chem. 48(8):1133-1137, 1976; Liu et al., Anal. Chem. 69:728A, 1997; Wyttenbach et al., Int. J. Mass Spec. 212:13, 2001; Hudgins et al., Int. J. Mass Spec. Ion Proc. 165/166:497-507, 1997; Wu et al., Anal. Chem. 70(23):4929-4938, 1998), time-of-flight (TOF) (Ruotolo et al., Int. J. Mass Spec. 219(1):253-267, 2002; Steiner et al., Rapid Comm. In Mass Spec. 15(23):2221-2226, 2001; Hoaglund et al., Anal. Chem. 70:2236-2242, 1998), quadrupole ion trap (QIT), (Liu et al., Anal. Chem. 69:728A, 1997; Creaser et al., Anal. Chem. 72(13):2724-2729, 2000), linear ion trap (LIT), (Sowell et al., J. Am. Soc. Mass Spectrom. 15:1341-1353, 2004), and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (Creaser et al., Anal. Chem. 72(13):2724-2729, 2000).
Typically, mass analysis is performed after an ion mobility separation. However, in some implementations the ability of QIT and LIT MS to trap and concentrate ions have been utilized as an ion mobility injection mechanism within low-field IMS instruments operating under near vacuum conditions (Liu et al., Anal. Chem. 69:728A, 1997; Creaser et al., Anal. Chem. 72(13):2724-2729, 2000; Sowell et al., J. Am. Soc. Mass Spectrom. 15:1341-1353, 2004). This union allows for higher IMS duty cycles to be achieved along with MS/MS experiments prior to mobility separation.
However, because of the vacuum requirement of ion trapping methodologies for concentrating ions prior to mobility analysis, these instruments have only been realized using low pressure (1-100 Torr) ion mobility analysis. Since IMS achieves a better resolution at higher pressures, IMMS systems employing a high pressure in the IMS segment are generally more desirable. Higher pressure IMMS have only been obtained using QMS (Karasek et al., Anal. Chem. 48(8):1133-1137, 1976; Wu et al., Anal. Chem. 70(23):4929-4938, 1998; Dugourd et al., Rev. Sci. Instrum. 68:1122-1129, 1997) and TOF (Steiner et al., Rapid Comm. in Mass Spec. 15(23):2221-2226, 2001) systems. The disadvantage to these instrumental configurations has been their inability to provide the valuable fragmentation information from MSn analyses.
Conventionally, a successful low-field IMMS instrument utilizes a mass analysis method capable of obtaining m/z information on a time scale much faster than the common signal averaged IMS experiment; however, this has not always been the case. Early IMMS unions used boxcar averaging with a mobility scanning technique that required two ion gates working in tandem to selectively filter ions for mass analysis (Karasek et al., Anal. Chem. 48(8):1133-1137, 1976). Despite being significantly slower than current methods, this ion mobility filtration technique has recently been reexamined, wherein a scanning ion mobility spectrometer was interfaced with a triple quadrupole mass spectrometer (Sysoev et al., Rapid Comm. in Mass Spec. 18(24):3131-3139, 2004). Using this technique it was possible to selectively filter ions prior to mass analysis, however, the nature of the experiment and approach excluded and precluded the possibility of collection of MSn data on low abundance analytes because, as appreciated and disclosed herein below by the present Applicant, there is no mechanism for the selective sequestration and fragmentation of analytes of interest. Moreover, the serial nature of the sampling results in an undesirably high MS duty cycle
Therefore, the art described above demonstrates the significant need for the development of novel and versatile platforms for multidimensional analytical separations that appreciate and incorporate elements for proper mating of process timescales.